Tat-LK15介导siRNA干扰大鼠脊髓背角nNOS表达对神经病理性疼痛的治疗作用

发布时间：2018-03-16 00:06

本文选题：Tat-LK　切入点：RNA　出处：《解放军医学杂志》2017年08期 　论文类型：期刊论文

【摘要】：目的探讨细胞穿透肽Tat-LK15介导siRNA干扰大鼠脊髓背角神经元型一氧化氮合酶(nNOS)表达对神经病理性疼痛的治疗效应。方法采用Tat-LK15介导FAM-siRNA转染原代大鼠脊髓背角神经元细胞(RN-dsc),于倒置荧光显微镜观察其转染效果。健康雄性SD大鼠50只,随机分为5组(n=10):正常组、假手术组、神经病理性疼痛组(SNL组)、Tat-LK15-nNOS siRNA组(TS组)和Tat-LK15-NC siRNA组(TN组)。采用脊神经结扎法(SNL)制作神经病理性疼痛模型,正常组不予任何处理,假手术组仅暴露脊神经;SNL组、TS组、TN组构建SNL模型同时鞘内置管,于第7天开始每天分别鞘内注射10μl生理盐水、10μl含5μg siRNA的Tat-LK15-nNOS siRNA和Tat-LK15-NCsiRNA,持续7d。建模前1d及建模后3、7、10、14d测定各组大鼠机械缩足反应阈值(PWMT)及热缩足反应潜伏期(PWTL)。第14天测定结束后取L4-6节段脊髓,采用q-PCR及Western blotting检测脊髓背角nNOS表达水平。结果倒置荧光显微镜观察显示,Tat-LK15可成功介导siRNA转染原代大鼠脊髓背角神经元。与假手术组比较,SNL组建模后第3天起PWMT降低、PWTL缩短,脊髓背角nNOS mRNA及蛋白表达上调(P0.01),正常组上述指标与假手术组比较差异无统计学意义;与SNL组比较,TS组第10、14天时PWMT增高、PWTL延长,脊髓背角nNOS mRNA及蛋白表达降低(P0.01),TN组上述指标与SNL组比较差异无统计学意义。结论 Tat-LK15可成功介导nNOS siRNA转染脊髓背角神经元,沉默nNOS基因的过度表达,对SNL大鼠神经病理性疼痛具有一定的治疗作用。
[Abstract]:Objective to investigate the therapeutic effect of cellular penetrating peptide (Tat-LK15) mediated by siRNA on the expression of neuronal nitric oxide synthase (NOS) in the spinal dorsal horn of rats. Methods Tat-LK15 mediated FAM-siRNA was used to transfect the primary spinal cord dorsal horn neurons into fine neurons. The transfection effect of RN-dscus was observed by inverted fluorescence microscope. The rats were randomly divided into 5 groups: normal group, sham-operated group, neuropathic pain group, Tat-LK15-nNOS siRNA group (TS group) and Tat-LK15-NC siRNA group (TN group). Spinal nerve ligation was used to make neuropathic pain model. In the sham operation group, only the spinal nerve was exposed to the SNL group. The TS group and the TN group were used to construct the SNL model and to place an intrathecal tube in the sheath at the same time. Tat-LK15-nNOS siRNA and Tat-LK15-NCsiRNAs containing 5 渭 g siRNA were injected intrathecally into the sheath of 10 渭 l saline 10 渭 l daily for 7 days from the 7th day onwards. The threshold of mechanical foot contraction and the latent period of thermal contraction were measured 1 day before modeling and 3 days after modeling. The latency of thermal contraction was measured on the 14th day. L4-6 segments of spinal cord were taken after completion. The expression of nNOS in the dorsal horn of spinal cord was detected by q-PCR and Western blotting. Results the results of inverted fluorescence microscope showed that Tat-LK15 could successfully mediate siRNA transfection into primary spinal dorsal horn neurons of rats. Compared with sham operation group, PWMT decreased the length of PWMT from the 3rd day after modeling. The expression of nNOS mRNA and protein in the dorsal horn of spinal cord was up-regulated (P 0.01). There was no significant difference in the above indexes between the normal group and the sham operation group, and the increase of PWMT in the TS group was longer than that in the SNL group on the 10th and 14th day after operation. The expression of nNOS mRNA and protein in the dorsal horn of spinal cord was decreased. There was no significant difference between the two groups. Conclusion Tat-LK15 can successfully transfect nNOS siRNA into spinal dorsal horn neurons and silence the overexpression of nNOS gene. It has certain therapeutic effect on neuropathic pain in SNL rats.
【作者单位】：江西省妇幼保健院麻醉科;广州军区广州总医院麻醉科;
【基金】：国家自然科学基金(81100817,81371233) 广东省自然科学基金面上项目(S2012010009271)~~
【分类号】：R402

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